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Abstract

Background: Transmission of HIV from mother to child through breast-feeding remains a global health challenge, particularly in developing countries. Breast milk from an HIV-infected women may contain both cell-free HIV-1 and cell-associated virus; however, the impact of human breast milk on HIV infection and replication in CD4+ cells remain poorly understood.

Objectives: In the present study, we evaluated the effects of breast milk in vitro on infection of CD4+ cells with cell-free HIV-1, including effects on HIV-1 receptor expression, reverse transcription, integration, and viral transcription. Additionally, we evaluated the ability of breast milk to inhibit cell-associated transmission of HIV-1 from infected CD4+ T lymphocytes.

INTRODUCTION

Mother-to-child transmission of HIV-1 through breast-feeding is a major public health concern in developing countries where HIV-infected women have limited access to antiretroviral treatment and/or safe alternatives to breast-feeding.1-3 Epidemiologic studies have shown varying rates of HIV breast milk transmission, with up to one third of HIV-positive women transmitting virus to their infants during breast-feeding.4,5 Breast-feeding infants born to HIV-infected women are less likely to become infected if they are exclusively breast-fed as compared with infants who receive other foods and fluids in addition to breast milk,6-9 supporting a protective role for breast milk in preventing HIV-1 infection in the infant. Both innate and adaptive immune factors with known antiviral activity, including macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated upon activation, normal T cell expressed and secreted (RANTES), stromal cell-derived factor-1 (SDF-1), and secretory immunoglobulin A (sIgA),10-16 have been identified in the breast milk of HIV-positive mothers. Whereas increased levels of RANTES in breast milk have been linked to a greater risk of vertical HIV-1 transmission during breast-feeding,12,16 other immune constituents including sIgA14,15 and α-defensins16 in breast milk do not seem to correlate with infant infection.

Elevated maternal viral load in breast milk is an accepted risk factor for transmission of HIV-1 from mother to infant during breast-feeding, and it is known that breast milk from HIV-positive mothers may contain both cell-free and cell-associated viruses.17-22 Increased levels of HIV-1 RNA in milk have been shown to correlate positively with virus transmission20; however, several studies have also suggested that cell-associated virus in milk may, in fact, be a more significant predictor of infant HIV-1 infection during breast-feeding.21-24 Infected cells in breast milk may include both CD4+ lymphocytes and macrophages,25-28 and recent studies have identified resting CD4+ lymphocytes as a potential cellular reservoir for HIV-1 in breast milk.26-28 Importantly, breast milk cells harboring HIV proviral DNA seem to be less responsive than cell-free HIV-1 RNA to short-term maternal antiretroviral (ARV) intervention during late pregnancy and early lactation.29-31

In the present study, we sought to determine the effect of human breast milk on cell-free HIV-1 infection and replication in CD4+ cells in vitro and on transmission of cell-associated HIV-1 from infected CD4+ lymphocytes. Our results indicate that breast milk contains innate factors that potently inhibit infection with cell-free HIV-1 in CD4+ target cells by blocking an early stage of the viral life cycle, but breast milk seems ineffective at preventing cell-associated transmission of virus from HIV-infected CD4+ T lymphocytes to susceptible target cells in vitro.

METHODS

Breast Milk

Breast milk was obtained by manual self-expression from 3 healthy HIV-negative donors, with established lactation at 1-month postpartum. Breast milk was obtained from either the left or right breast up to 2 hours after the infant had last breast-fed from that side. Whole breast milk was centrifuged to separate the lipid and cellular fractions from the skim milk. All experiments were performed using the skim milk fraction from individual donors; breast milk was not pooled. The skim milk was filtered through a 0.2-μm cellulose acetate filter and stored in aliquots at −80°C before use. Protocols for this study were approved by the Dartmouth Committee for the Protection of Human Subjects, and informed consent was obtained from all donors before collecting breast milk.

CD4+ TZM-bl cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (contributed by Dr. John C. Kappes, Dr. Xiaoyun Wu, and Tranzyme, Inc). The cells were grown and maintained in Dulbecco's Modified Eagle's Medium (Gibco/Invitrogen) supplemented with 10% FBS. TZM-bl cells stably express CD4, CCR5, and CXCR4 and contain reporter genes for both firefly luciferase and β-galactosidase under control of the HIV-1 long terminal repeat (LTR) promoter. In the present study, luciferase expression was used to quantify HIV-1 infection.

HIV-1 Isolates

Clade B primary HIV-1 isolates used for this study were kindly provided by Dr. John Moore (Weill Medical College of Cornell University, New York, NY) and include HIV-1CM235 (R5 tropic), HIV-1HC4 (X4 tropic), and HIV-1C7/86 (X4/R5 tropic). In addition, HIVBaL (R5 tropic) was used for certain experiments (National Institutes of Health AIDS Research and Reference Reagent Program). All HIV-1 isolates were propagated in PHA-activated PBMCs and titered using TZM-bl cells.

HIV-1 Infection of PBMCs

PHA-activated PBMCs were resuspended at a density of 2 × 106 cells per milliliter in breast milk diluted to a final concentration of 1:5 in RPMI/10% FBS. The effect of breast milk from each of the 3 donors was evaluated using PBMCs from individual donors. The cells were then inoculated with 100 TCID50 of either HIV-1C7/86 or HIV-1BaL for 2 hours at 37°C. After the 2-hour incubation, PBMCs were washed and resuspended in either fresh media or replenished breast milk and plated in triplicate wells of a 24-well plate. HIV-1 replication was assessed by enzyme-linked immunosorbent assay for viral p24 antigen in culture supernatants on days 3, 5, and 7 after infection (PerkinElmer, Boston, MA). Controls included PBMCs infected with HIV-1 in the absence of breast milk and PBMCs that were not exposed to HIV-1. In other experiments, PBMCs were lysed after 48 hours and genomic DNA was extracted and used for real-time polymerase chain reaction (PCR) quantification of HIV-1 reverse transcription and integration as described below.

Coculture of HIV-Infected CD4+ T Lymphocytes

To assess the ability of breast milk to decrease cell-associated HIV-1 infection, HIV-infected CD4+ T lymphocytes were cocultured directly with uninfected TZM-bl cells and the levels of luciferase activity quantified as an indicator of HIV-1 infection. CD4+ T lymphocytes were first enriched from PBMCs as described and infected with 100 TCID50 of HIV-1BaL for 5 days at 37°C before use. On the day of the experiment, the lymphocytes were washed with fresh media to remove cell-free virus. In parallel, TZM-bl cells were seeded into the wells of a 96-well plate (105 cells/well) and allowed to adhere overnight. Breast milk at a 1:5 final dilution or media was then added to designated wells of TZM-bl cells, followed by the addition of washed HIV-infected CD4+ T lymphocytes at a final density of 1 × 105 lymphocytes per well. To control for the release of cell-free HIV-1 from the infected lymphocytes during coculture, an equivalent number of HIV-infected CD4+ lymphocytes (1 × 105 lymphocytes/well) was seeded into wells with no TZM-bl cells and the cell-free virus was collected in the supernatant after 30 hours. This supernatant was then added to TZM-bl cells and used to assess the effects of breast milk against cell-free HIV-1. Additional controls included TZM-bl cells cocultured with uninfected CD4+ T lymphocytes. After 30 hours, the cells were lysed directly in the wells with Beta-Glo reagent (Promega, Madison, WI) and luciferase activity was quantified on an LMaxII384 luminometer (Molecular Devices, Sunnyvale, CA).

Statistical Analysis

RESULTS

Breast Milk Inhibits Cell-Free HIV-1 Infection of CD4+ Cells

The effects of breast milk on HIV-1 infection were first analyzed with CD4+ TZM-bl cells using LTR-driven luciferase expression as a quantitative assessment of viral infection (Fig. 1). Cell-free HIV-1 was added to TZM-bl cells in the presence of 5-fold serial dilutions of breast milk, and the results were compared with media controls. Breast milk dilutions were started at 1:4 as higher concentrations of milk were found to be cytotoxic to TZM-bl cells after 24-48 hours in culture (data not shown). Our results demonstrate that breast milk inhibits cell-free HIV-1 infection in a dose-dependent manner, with significant decreases at breast milk dilutions of 1:4 and 1:20 as compared with media controls (P = 0.03) (Fig. 1A). In the absence of HIV-1, breast milk alone did not induce expression of luciferase in the treated TZM-bl cells over the range of concentrations tested (Fig 1A). Inhibition of cell-free HIV-1 by breast milk was not due to loss of TZM-bl cell viability (CellTiter 96 AQueous One; Promega) (data not shown).

Breast Milk Decreases the Cell Surface Expression of CCR5 but Not CD4 and CXCR4 on PBMCs

To determine whether exposure to breast milk alters cell surface levels of HIV-1 receptors and/or coreceptors, both freshly isolated and PHA-activated PBMCs were analyzed by FACS for expression of CD4, CXCR4, and CCR5 after incubation for 30 minutes with breast milk (Fig. 2). Expression of CD4 was similar among both freshly isolated and PHA-activated cells, and no differences in CD4 expression were observed among cells treated with either breast milk or media (Fig. 2A). PHA activation significantly increased surface expression of CXCR4 as compared with freshly isolated cells (Fig. 2B); however, levels of CXCR4 expression were again similar for cells treated with either breast milk or media, indicating that short-term exposure to breast milk does not significantly alter CXCR4 surface expression on either activated or nonactivated cells. Overall, we found the expression of CCR5 on both freshly isolated and PHA-activated PBMCs to be extremely low, even upon repeat experiments. We did note a decrease in detectable CCR5 in the presence of breast milk as compared with media (Fig 2C). Although suggestive, these results are consistent with reports of naturally occurring anti-CCR5 antibodies found at high frequency in the breast milk of both HIV-negative and HIV-positive women that may block interaction of R5-tropic HIV-1 strains through interaction with the CCR5 coreceptor.36

Breast Milk Inhibits Early Events in the HIV-1 Life Cycle

To assess the impact of breast milk on HIV-1 postentry events, PHA-activated PBMCs were exposed to cell-free HIV-1BaL for 2 hours in the presence of breast milk, washed, and evaluated 48 hours later for the products of HIV-1 reverse transcription and integration by quantitative real-time PCR (Fig. 3). These experiments revealed a significant inhibition of HIV-1 reverse transcription in PBMCs infected with cell-free HIV-1 in the presence of breast milk relative to media controls (P = 0.004) (Fig. 3A, breast milk/media). When PBMCs were maintained in the presence of breast milk throughout the 48-hour incubation period, the inhibitory effect of breast milk on HIV-1 reverse transcription was even more pronounced (P = 0.001) (Fig. 3A, breast milk/breast milk).

Breast milk inhibition of postentry events was also observed at the level of HIV-1 integration in PBMCs exposed to breast milk during infection (Fig. 3B). Levels of integrated proviral DNA at 48 hours were significantly lower in PBMCs infected with HIV-1 during a 2-hour exposure to breast milk as compared with media controls (P = 0.02) (Fig. 3B, breast milk/media). This inhibition was markedly enhanced when the cells were maintained in the presence of breast milk for the full 48-hour period after infection with HIV-1 (P = 0.008) (Fig. 3B, breast milk/breast milk).

Effect of Breast Milk on HIV-1 Transcription and p24 Release

PBMCs infected with HIV-1 in the presence of breast milk were evaluated on days 3, 5, and 7 after infection for the early virus transcripts tat and rev (Fig. 4) and for release of p24 antigen into the culture supernatants (Fig. 5). In PBMCs that were maintained in breast milk for the duration of the experiment, there was a significant reduction in HIV-1 transcription at day 3 after infection (P < 0.001), which was subsequently lost by days 5 and 7 (P = 0.06). In some cases, as shown, breast milk enhanced the level of HIV-1 transcripts measured on day 5 after infection, but this effect was not seen by day 7 of culture (Fig. 4).

Analysis of p24 levels present in the culture supernatants of HIV-1-infected PBMCs showed no significant difference in the levels of HIV-1 p24 released into the culture supernatant among cells continuously treated with breast milk at days 3, 5, and 7 after infection as compared with media controls (Fig. 5). Taken together, these results suggest that breast milk exerts a significant inhibitory effect on the early stages of infection with cell-free HIV-1, but this inhibitory effect is not sustained over prolonged culture, even with continuous exposure of target cells to breast milk. Treatment of cells with breast milk did not reduce release of HIV-1 p24 into the culture supernatants nor did it decrease spread of the virus in the culture.

Breast Milk Is Ineffective at Blocking Cell-Associated Infection With HIV-1

To investigate whether breast milk impacts cell-associated infection with HIV-1, CD4+ T lymphocytes were infected with HIV-1 and cocultured with uninfected CD4+ TZM-bl target cells in the presence of breast milk. Transmission of virus from the infected CD4+ T lymphocytes was measured by assessing induction of luciferase expression in the recipient TZM-bl cells. As a control for release of cell-free HIV-1 from the infected lymphocytes during the coculture period, equivalent numbers of HIV-infected CD4+ T lymphocytes were cultured in wells without TZM-bl cells, and the resulting supernatants containing cell-free HIV-1 were then added to TZM-bl cells in the presence of breast milk.

The results of this experiment confirm that breast milk is effective at blocking cell-free HIV-1 infection, using only the supernatants from the infected CD4+ T lymphocytes as a source of cell-free virus (Fig. 6). However, when infected CD4+ T lymphocytes were cocultured directly with TZM-bl cells in the presence of an equivalent concentration of breast milk, no inhibition of HIV-1 infection was observed. Control cocultures containing uninfected CD4+ T lymphocytes showed no induction of luciferase activity in TZM-bl cells (Fig 6), indicating that the lymphocytes alone were not activating the target cells either directly through cell-cell interactions or indirectly via secretion of soluble mediators. The results of these experiments support the concept that innate factors in human breast milk are relatively ineffective at blocking cell-associated HIV-1 as compared with cell-free virus infection of CD4+ target cells.

DISCUSSION

Human breast milk is a highly complex fluid that contains several direct-acting antimicrobial agents, such as lactoferrin,37 and additional immunomodulatory factors, such as cytokines and chemokines.38 CD4+ and CD8+ T lymphocytes39,40 and HIV-specific antibodies, notably sIgA,13-15 have also been identified in breast milk from HIV-positive women, indicating that both innate and acquired immunological components are present in breast milk and may contribute to conferring protection against HIV-1 transmission to the infant. Elevated breast milk viral load positively correlates with HIV-1 transmission to the infant; however, the relative contribution of cell-free and cell-associated HIV-1 to vertical transmission is unclear and may be influenced by underlying conditions in the mother, such as mastitis,24 and by administration of short-term maternal ARV therapy at the time of pregnancy, delivery, and/or lactation.29-31

In the present study, we sought to determine the effect of human breast milk on cell-free HIV-1 infection and replication in CD4+ cells in vitro and on cell-associated transmission of HIV-1 from infected CD4+ T lymphocytes. Our results demonstrate potent inhibition of cell-free HIV-1 by breast milk during the early stages of virus infection of CD4+ cells but little or no inhibition of cell-associated HIV-1 infection of susceptible target cells. These findings have implications for understanding the potential role of innate factors in protection against HIV-1 infection and transmission of virus to the infant during breast-feeding.

Breast milk from HIV-1-negative donors was used in this study to examine the contribution of innate factors in preventing HIV-1 infection. Chemokines such as RANTES and SDF-1, which function as ligands for CCR5 and CXCR4, respectively, have been identified in human breast milk; however, it is unclear whether these factors act in the milieu of breast milk to block binding of cell-free HIV-1 to susceptible target cells and/or whether they affect HIV-1 infection and replication within the localized environment of the mammary gland. Levels of MIP-1β and RANTES in breast milk were found to be higher among a cohort of HIV-1-infected Kenyan women as compared with uninfected women, and transmission of HIV-1 to the infant was positively associated with increased RANTES in the breast milk,12-16 suggesting that RANTES itself does not play an inherently protective role against HIV-1 infection in the infant.

Although we found a modest decrease in CCR5 levels on CD4+ PBMCs exposed to breast milk, inhibition of cell-free HIV-1 by breast milk was shown to be independent of HIV-1 tropism, making it unlikely that the inhibitory activity is due to a blockade of specific virus-coreceptor interactions. Rather, our results suggest the presence of broadly active nonspecific factors that may block viral infection, possibly by promoting HIV-1 aggregation, decreasing interaction of HIV-1 with CD4, and/or inhibiting the early stages of CD4-mediated viral entry. Sulfated glycolipids and glycosaminoglycans,41 mucin,42,43 and certain long-chain polyunsaturated fatty acids44 may fulfill such a role and have been identified in human breast milk. These factors are known to have antiviral activity against HIV-1 in vitro,41-43 and increased concentrations of fatty acids in the breast milk of HIV-positive women have been associated with reduced transmission of HIV-1 to the infant during breast-feeding.44

Although we did not attempt to quantify the levels of nonspecific factors in the breast milk used in this study, we did find that the inhibitory activity of the breast milk was maintained at dilutions of 1:20 and higher, suggesting a relatively abundant factor and/or a potent inhibitory effect on cell-free HIV-1. It should be noted that all our experiments were performed using the skim fraction of the breast milk, in which the lipids were removed, and therefore, our results do not preclude the activity of factors such as mucin and certain other proteoglycans that may be associated with milk fat globules.41-43 However, our findings do support the presence of innate factors in the skim fraction of human breast milk that are able to decrease early events in the infection of CD4+ cells with cell-free HIV-1.

By comparison, we found that the inhibitory activity of breast milk against HIV-1 was not sustained over prolonged in vitro culture with CD4+ PBMCs, despite continuous replenishment of fresh breast milk in the culture. This finding was unexpected given the potent inhibitory effect of breast milk on cell-free HIV-1, and we reasoned that this lack of sustained inhibition might be due to direct cell-cell spread of the virus in culture. To evaluate this possibility, we established cocultures of HIV-infected CD4+ T lymphocytes with CD4+ indicator cells in the presence of breast milk at concentrations previously shown to inhibit cell-free HIV-1. We found that breast milk was ineffective at blocking HIV-1 transmission from infected CD4+ lymphocytes when the cells were in direct contact with uninfected target cells. Use of supernatants from the infected CD4+ lymphocytes yielded cell-free HIV-1, and this infection was again inhibited by breast milk. In our experiments, breast milk was added to the cultures before the addition of infected lymphocytes, suggesting that even preexposure of susceptible target cells to breast milk is insufficient to block cell-cell interactions as compared with virus-cell interactions under similar conditions.

Transmission of HIV-1 to the breast-feeding infant likely involves interaction of HIV-1-infected breast milk cells with mucosal surfaces in the infant's oral, nasopharyngeal, and/or gastrointestinal tract. Contact between HIV-1-infected cells and HIV-1-uninfected epithelial cells is postulated to occur by way of a “virally mediated synapse” with binding of the HIV-1 envelope glycoproteins to the epithelial glycosphingolipid receptor galactosyl ceramide (GalCer) and stabilization of the interaction with integrin- and agrin-dependent engagement.45 Mucosal sIgA specific for the ELDKWA epitope of HIV-1 gp41 has been shown to inhibit this process by blocking viral transcytosis across epithelial cells. sIgA and IgG antibodies specific for HIV-1 gp160 have been identified in the colostrum and milk of HIV-positive mothers; however, the in vitro functionality of these antibodies to block HIV-1 transcytosis is similar among transmitting and nontransmitting mothers.15 This suggests that the presence and/or specificity of naturally occurring antiviral antibodies in breast milk may be inadequate to prevent cell-mediated transcytosis of HIV-1 across the infant intestinal epithelium. Our results further suggest that innate factors in human milk, which seem to be effective in blocking cell-free HIV-1 infection, may be less effective at blocking cell-cell interactions and may therefore be less likely to play a protective role in preventing cell-associated HIV-1 transmission to the breast-feeding infant.

Recent studies have identified a large pool of latently infected resting CD4+ lymphocytes in the breast milk of HIV-positive mothers, which represent more than 90% of the viable breast milk cells.26-28 These cells seem to have a greater capacity to replicate HIV-1 upon in vitro activation as compared with peripheral blood CD4 cells,28 raising the possibility that a significant cellular reservoir for HIV-1 exists in the breast milk of HIV-infected women. To date, short-term ARV therapy initiated during late pregnancy and/or early lactation has not been effective at reducing the levels of HIV-1 proviral DNA in breast milk,29,30 in contrast to HIV-1 RNA levels that decline rapidly in the milk, suggesting that the cellular reservoir for HIV-1 in breast milk may be more refractory to treatment.27 Breast milk cells have also been shown to remain viable for extended periods in vitro in the presence of breast milk, whereas cell-free HIV-1 is progressively inactivated in a time-dependent fashion by exposure to human milk.46

Taken together with our present findings, these results suggest that HIV-infected resting CD4+ T lymphocytes may form a significant and persistent reservoir for HIV-1 in breast milk that may be unresponsive to short-term ARV intervention,30 and these cells may successfully transmit HIV-1 infection to susceptible target cells even in the presence of innate factors in the breast milk that can block cell-free virus. Whether similar findings also apply to breast milk from HIV-positive mothers is currently under investigation in our laboratory. Little is known about how HIV-1 infection in the mother alters the composition of innate factors present in breast milk and whether nutrition and other factors may impact the efficacy of breast milk to inhibit HIV-1 transmission to the infant. It is reasonable to assume that ARV interventions aimed at preventing mother-to-child breast milk HIV-1 transmission must impact both the cell-free and cell-associated viruses in this compartment, and therefore, the rational design and implementation of such interventions remain a high priority.

ACKNOWLEDGMENTS

We extend our sincere thanks to the women who consented to donate breast milk for use in this study and to Dr. Susana Asin for laboratory support, many helpful discussions, and assistance in preparation of this article.

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